Project Summary/Abstract Cocaine use disorder is a public health concern with no effective pharmacotherapies, despite clear need. Even though both males and females become addicted to cocaine, females transition to addiction faster, have more trouble quitting cocaine and relapse more frequently than men. Many of these features have been captured in preclinical models of addiction where female nonhuman primates and rodents take more drug, work harder to obtain drug and are more sensitive to cue- and stress-primed reinstatement. Studies have shown that the increased cocaine-taking and drug craving fluctuates with the menstrual cycle (estrous cycle in rodents) where consumption and craving are most evident during estrus (when ovarian hormone levels are high). Other evidence shows that the ventral tegmental area (VTA) to nucleus accumbens (NAc) dopamine (DA) neurons are more active at baseline and release more DA in response to phasic stimulation during this same period. While there have been numerous studies focusing on defining the causal link between specific hormones and alterations in dopamine system function, understanding how the intact estrous cycle drives differences in cocaine reinforcement is critical to understanding sex differences in vulnerability. Here we aim to understand how the VTA to NAc DA system is altered in intact cycling females over the estrous cycle to change reinforcement for cocaine. In this proposal, we will use a combination of cutting-edge techniques such as pathway-specific optogenetic stimulation and designer receptors exclusively activated by designer drugs approaches in vivo to isolate the VTA to NAc DA neurons and understand the effect of the estrous cycle on the function of these DA neurons and the effect this has on cocaine-taking behavior. We will then use ex-vivo slice voltammetry to precisely understand the mechanisms involved in the increased DA functioning during estrus. We will determine if the effect is happening at the level of local circuits within the NAc, DA synthesis, or changes in the readily releasable pool of DA. These experiments will elucidate the effect of the estrous cycle on the VTA to NAc DA pathway and will provide new information about biological factors that increase vulnerability to drug addiction. This will provide new targets for pharmacotherapy development as we learn more about how to modulate the DA system in ways that will decrease cocaine-taking behavior without producing addictive or aversive effects.